Pluripotent inner cell mass (ICM) cells of mammalian preimplantation embryos comprise the earliest culturable stem cells from mammalian organisms and are unusual in a number of respects. When introduced into a histocompatible host, such cells can either form a malignant teratocarcinoma, or participate in normal developmental processes depending on their sites of placement. As represented by embryonal stem (ES) or embryonal carcinoma (EC) cells, pluripotent stem cells demonstrate unique patterns of DNA methylation, promoter usage and transcriptional regulation. In previous investigations, we characterized a stem cell- specific silencer which represses expression from a variety of promoters and ultimately leads to methylation of linked transcription units in Ec cells. Recently, we identified a nuclear factor, binding factor A, which binds specifically to the repressor binding site (RBS) sequence. We also have isolated novel stem cell cDNA clones encoding RBS- and other DNA- binding proteins. The factor and/or clones we have identified may be master regulators, crucial in the maintenance of the transformed state of undifferentiated stem cells, or in the control of early mammalian development. At the very least, they are probable participants in the regulatory network of differentiating mammalian cells. Our specific aims, which focus on mechanisms of gene regulation in mouse embryos, and in EC and ES cells, are as follows: l. To purify and characterize the putative repressor factor, binding factor A, and other factors which bind specifically to the stem cell repressor binding site (RBS). 2. To characterize stem cell cDNA clones encoding the four DNA-binding proteins, lB/C, 23B, 4D, and 20B. 3. To monitor RBS-induced repression and methylation of transcriptional units in human and murine EC cells, differentiating murine ES cells, and preimplantation mouse embryos.